For diagnosis and control of diabetes, measurement of glycated proteins is extremely important. In particular, glycated albumin and hemoglobin A1c are used in many cases as indexes essential at the scene of medical treatments. The glycated albumin and hemoglobin A1c have been quantified by quantitative assays such as electrophoresis, ion-exchange chromatography, affinity chromatography, and immunization. In recent years, attempts have been made to develop an enzymatic method which enables a large-scale treatment of a large amount of a sample in a simpler manner (Non-patent Documents 1 and 2).
An example of the best known enzymatic method is a method of measuring a glycated amino acid or a glycated peptide which is generated from digestion of a glycated protein in a sample by a protease. However, a reaction of the protease is slow in general, and it was difficult for the digestion of the protein to reach 100% digestion in a short period of time even though a large amount of the protease is used. In addition, generated hydrogen peroxide can be quantified through color development of a colorant by using a peroxidase. However, the colorant, in particular, one of a leuco-type has a problem in that stability thereof is not good.
There are known methods using, as additives for promoting a reaction of the protease by denaturing a protein that is a measurement target at the time of the measurement of the glycated protein, tetrazolium salts (Non-patent Document 2 and Patent Documents 1 to 7) and sulfonic acid compounds and/or nitro compounds (Patent Documents 8 and 9). However, a tetrazolium salt strongly reacts with a reductive compound such as the glycated protein or ascorbic acid and develops color, so the tetrazolium salt causes an abnormal value in a sample containing the glycated protein in a high concentration or a sample containing ascorbic acid. In addition, many of the sulfonic acid compounds are inhibitors of an enzyme. In particular, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, lithium lauryl sulfate, and the like which are often used are such strong inhibitors that they serve as reaction terminators of an enzyme which acts on the glycated amino acid and/or the glycated peptide. Further, the nitro compound is colored so that a reagent blank has an increased value, resulting in a decrease in measurement accuracy.
As described above, the “additives for promoting a reaction of the protease by denaturing a protein that is a measurement target” which are currently used are not satisfactory.
In addition, as a case where a leuco-type colorant is used in measurement, there is known measurement of hemoglobin A1c by using a reagent to which N-(carboxymethylaminocarbonyl)-4,4-bis(dimethylamino)biphenylamine (hereinafter, sometimes referred to as “DA64”) (Non-patent Document 2) is added. However, there is no description regarding stability and preservation of the reagent. There has not been known a reagent containing a leuco-type colorant which has long-term stability in a liquid state.    Patent Document 1: WO 02/27012    Patent Document 2: WO 02/27330    Patent Document 3: WO 2002/027331    Patent Document 4: JP-A-2000-93199    Patent Document 5: JP-A-2001-292795    Patent Document 6: JP-A-2003-232789    Patent Document 7: WO 2000/210100    Patent Document 8: WO 2004/007760    Patent Document 9: WO 03/107011    Non-patent Document 1: Kouzuma et. al., “An enzymatic method for the measurement of glycated albumin in biological sample”, Clinica Chimica Acta, 2002, 324, p. 61-71    Non-patent Document 2: Ikunosuke Sakurabayashi et. al., “New enzymatic assay for glycated hemoglobin”, Clinical Chemistry, 2003, 49, 2, p. 269-274